Heat-inducible expression system for a foreign gene in cultured tobacco cells using the HSP18.2 promoter of Arabidopsis thaliana

Abstract

A system for the controlled expression of a foreign gene in cultured tobacco cells (Nicotiana tabacum, BY2) by temperature shift was constructed. A 925-base-pair (bp) DNA fragment containing the 5'-flanking region of a low-molecular-mass heat-shock protein gene (HSP18.2) of Arabidopsis thaliana was inserted upstream of the beta-glucuronidase reporter gene (GUS). The resulting HSP18.2-GUS construct was introduced into BY2 cells by electroporation or Agrobacterium-mediated transformation. Transient expression of the HSP18.2 promoter in protoplasts was very low regardless of the heat shock. Although expression of the HSP18.2-GUS chimeric gene in the stable transformants of BY2 was hardly detected in culture at 25 degrees C, the expression increased rapidly on the transcriptional level when the incubation temperature was shifted to 35-37 degrees C. The optimal temperature for heat-shock induction was 37 degrees C. After a 2-h incubation at 37 degrees C, GUS activity was about 1000-fold greater than that before heat shock. The amount of GUS mRNA was maximum 2 h after heat shock, and then decreased gradually.

Metabolic engineering of cultured tobacco cells

“…To express a foreign gene efficiently in cultured cells, a good expression system using an appropriate promoter and ciselements is necessary. We reported that a promoter of heatshock protein gene (HSP18.2) of Arabidopsis thaliana, induced expression of the ␤-glucuronidase (GUS) reporter gene by heat treatment for 2 h at 37°C in BY2 cells (Yoshida et al, 1995). Similar results were observed in cultures in 500 mL flask and 3 L jar fermentor as reported here.…”

“…Cells of N. tabacum L. cv BY2 transformed with binary plasmid pGA482 containing the HSP18.2-GUS fused gene and NPTII gene (Yoshida et al, 1995;Shinmyo et al, 1996) were incubated in 95 mL of modified LS medium (Nagata, 1987) containing 100 g/mL kanamycin in 500 mL flasks, in the dark, at 130 rpm and 25°C 30 mL culture broth. After 7 d, the mixture was transferred to 3 L jar fermentor (Marubishi Bioengineering Co. Ltd., Tokyo, Japan) equipped with a four-turbine blade containing 1.5 L of the same medium, and cultured by agitation at 60 rpm and aeration of 0.6 vvm at 26°C in the dark.…”

BY‐2 Cells: Culture and Transformation for Live Cell Imaging

“…Besides chemical inducers, gene expression in the BY‐2 cell system may be induced by heat shock. In this respect, the heat‐sensitive promoter HSP18.2 may be used as an alternative to chemical inducers with fast kinetic responses (Yoshida et al, ; Shinmyo et al, ).…”

Conditional, recombinase‐mediated expression of genes in plant cell cultures

“…The HSP18.2 promoter has been shown to be strongly inducible in transgenic Arabidopsis plants (Matsuhara et al ., 2000; Takahashi et al ., 1992) and cultured tobacco BY‐2 cells (Yoshida et al ., 1995); it has also been utilized to control the expression of the CRE recombinase in genetic mosaic analysis of flower development in Arabidopsis (Sieburth et al ., 1998). The HSP18.2 promoter is optimally induced by a heat‐shock treatment at 37°C for 2 h (Yoshida et al ., 1995). Furthermore, this promoter was chosen to drive expression of the recombinase because the constitutive expression of CRE in plants had resulted in phenotypic aberrations (Coppoolse et al ., 2003).…”